3.4 Stability studies of PEG-FGF2 conjugates
To verify whether site-specific PEGylation improves the stability of FGF2 conjugates, SD rat plasma and mice wound fluid were used.
Compounds 5 , 6, and 7 were first incubated with rat plasma over 24 h and compared with FGF2 incubation. Solid-phase extraction was conducted at different time points, and the remaining FGF2 was analyzed using ELISA Kit to examine plasma stability (Figure 5A ). The results showed rapid degradation of native FGF2 in plasma, with its concentration decreased to less than 10% of the initial value within 30 min and complete degradation within 1 h at 37 °C. In contrast, all PEG-FGF2 conjugates showed enhanced stabilities relative to the unmodified FGF2. After 2 h of incubation in plasma, the residual FGF2 percentages of compounds 5 , 6, and7 were about 66%, 93% and 90% of the initial concentrations, respectively. After 8 h of incubation, about 33%, 76%, and 58% of the initial concentrations remained for compounds 5 , 6,and 7 , respectively. The data indicate that site-specific PEGylation can significantly improve the stability of FGF2 in plasma.
Acute wound fluid is generally rich in metalloproteinases (MMPs), especially MMP2 and MMP9 subtypes, within the initial 3 days after scald (Xu et al. , 2018), which may result in elevated levels of proteases that can rapidly degrade exogenous growth factors (Ladwiget al. , 2002). Therefore, the stability of PEG-FGF2 conjugates was further quantitatively analyzed by ELISA Kit after incubation in wound fluids collected within 1 day after scald (Figure 5B ). As expected, FGF2 mixed with wound fluid was degraded rapidly, for a remaining FGF2 rate of 4.47 ± 0.95% after incubation in wound fluid for 24 h. Compounds 5 , 6, and 7 were more stable, with remaining FGF2 rates of 32.00 ± 7.94%, 63.33 ± 3.51%, and 49.33 ± 5.13% respectively. These results indicate that all PEG-FGF2 conjugates exhibit obviously increased stability in wound fluids compared to unmodified FGF2.
Taken together, these results demonstrate that site-specific PEGylation can effectively protect FGF2 from degradation for increased stability. Among the tested PEG-FGF2 conjugates, compound 6 exhibited the strongest stability.